Field of the Invention
The present invention relates to a novel method for detection of target nucleic acid sequences by cyclic exonucleolytic reactions (CER) or exonucleolytic reactions (ER) using single-labeled immobilized probes on a solid phase.
Description of the Related Art
Microarray-based technologies are highlighted as promising tools for analyzing the presence, level or expression patterns of a gene or a gene population (Schena et al., 1995. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray, Science, 270:467-470; DeRisi et al., 1996, Use of a cDNA Microarray to Analyse Gene Expression Patterns in Human Cancer, Nature Genetics 14:457-460). However, since the conventional DNA microarray methods depend mostly on hybridization to detect target nucleotide sequences, they have serious shortcomings of a high rate of false positives. In particular, cross reactions (non-specific hybridizations) usually occurring in the conventional DNA microarrays and decreased sensitivity give rise to a sharp decrease in reliability of final hybridization signals (William E. Bunney, et al. 2003. Microarray Technology: A Review of New Strategies to Discover Candidate Vulnerability Genes in Psychiatric Disorders, Am. J. Psychiatry 160:4, 657-666).
To overcome such problems of microarray-based technologies, a variety of novel microarray approaches have been suggested depending on not only hybridization but also enzymatic reactions such as DNA polymerase reactions or ligase reactions for elevating reliability of target detection.
The single base extension (SBE) or minisequencing method (SBE: Shumaker et al. Mutation detection by solid phase primer extension. Hum. Mutat. 7:346-354 (1996); Pastinen, et al., Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res. 7:606-614 (1997)) employs 5′ to 3′ DNA polymerases in the presence of single-labeled ddNTPs to induce a single base extension of primers with single-labeled ddNTPs for target detection.
The ligation method (Affymetrix, INC, Enzymatic methods for genotyping on arrays, U.S. Pub. No. US2008/0131894) detects target sequences by ligation between array probes and single-labeled probes using ligases.
Although these conventional methods provide microarray-based target detection approaches using a single label molecule, these methods have serious shortcomings. For example, the SBE method and the ligation method require additional components, labeled ddNTPs and second labeled probes, respectively, and require additional reactions for these additional components. In addition, these methods are very likely to generate false positive signals due to inherent reaction error rates associated with DNA polymerases and ligases.
In the meantime, a solid phase-based TaqMan probe method has been reported to detect target sequences using 5′ to 3′ nuclease activities of DNA polymerases and dual-labeled probes (Liu et al., TaqMan probe array for quantitative detection of DNA targets, Nucleic Acid Res. 34:e4 (2006)). In the method, the dual-labeled probes are immobilized on a solid substrate. The method detects target sequences by measuring increase in signals from cleavage of dual-labeled probes by primer-dependent 5′ to 3′ nuclease activities of DNA polymerases. The primer-dependent 5′ to 3′ nuclease activities means that the dual-labeled probes are cleaved only by DNA polymerases bound to upstream primers or extended products of upstream primers.
This dual-labeled method possesses quite troublesome consideration factors. For example, the positions of reporter and quencher molecules on probes have to be determined by considering separation of two label molecules by enzymatic cleavage and remaining of reporter molecules on solid substrates even after the cleavage reactions. Upon increasing the distance between reporter and quencher molecules, the background signals are more likely to be generated because of unstable quenching. These tricky considerations associated with dual-labeled systems on probes are responsible for limitations and difficulties in probe design for the solid phase-based TaqMan probe method. Furthermore, the utilization of two label molecules is unfavorable in view of cost-effectiveness. In addition to this, the requirement for additional upstream primers for cleavage reactions makes this method to be worse.
Therefore, there remain long-felt needs in the art to develop novel DNA microarray technologies for detecting a target sequence, preferably a multiple of target sequences on a microarray by using only a single label molecule in a more convenient, reliable and reproducible manner. Furthermore, a novel real-time microarray method using only a single label molecule is also needed in the art for quantitative analysis of target nucleic acid sequences.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.